In general, a biosensor is capable of identifying biomolecules such as polynucleotides and polypeptides. One method includes introducing a biomolecule sample containing target biomolecules to a substrate having probes deposited thereon. The probe can include various biomolecules that can interact with target biomolecules, while the substrate can be a solid surface such as silica, surface-derivatized glass, polypropylene, and activated polyacrylamide. The target biomolecules can then interact (e.g., bond or hybridize) with one or more of the probes. Subsequently, the interaction can be analyzed based on the presence and location of the target biomolecule-probe interaction, which can be determined by one or more detection techniques such as optical, radiochemical, and electrochemical techniques.
Another method includes introducing a substrate having target biomolecules disposed thereon to a solution that can include complimentary polynucleotide or polypeptide probe samples derived from biomolecules that have been tagged with fluorescent dyes. The probe material interacts selectively with target biomolecues only where complimentary bonding sites occur. In other words, probe biomolecules with similar chemical characteristics (e.g., nucleotide sequence or amino acid sequence) to the target biomolecule interact with the target molecules, while dissimilar probe biomolecules do not significantly interact with the target biomolecules. Thereafter, the presence and quantity of bound probe biomolecules can be detected and analyzed in a manner similar to the detection techniques discussed above.
In regard to detection of target biomolecules, the ability to detect and identify trace quantities of chemicals has become increasingly important in virtually every scientific discipline, ranging from part per billion analyses of pollutants in sub-surface water to analysis of cancer treatment drugs in blood serum. Surface-enhanced Raman spectroscopy (SERS) has proven to be one of the most sensitive methods for performing such chemical analyses by the detection of a single molecule. (Nie, S. and S. R. Emory, “Probing Single Molecules and Single Nanoparticles by Surface Enhanced Raman Scattering”, Science, 275,1102 (1997)). A Raman spectrum, similar to an infrared spectrum, includes a wavelength distribution of bands corresponding to molecular vibrations specific to the sample being analyzed (the analyte). In the practice of Raman spectroscopy, the beam from a light source, generally a laser, is focused upon the sample to thereby generate inelastically scattered radiation, which is optically collected and directed into a wavelength-dispersive or Fourier transform spectrometer in which a detector converts the energy of impinging photons to electrical signal intensity.
Historically, the very low conversion of incident radiation to inelastic scattered radiation limited Raman spectroscopy to applications that were difficult to perform by infrared spectroscopy, such as the analysis of aqueous solutions. It was discovered in 1974, however, that when a molecule in close proximity to a roughened silver electrode is subjected to a Raman excitation source, the intensity of the signal generated is increased by as much as six orders of magnitude. (Fleischmann, M., Hendra, P. J., and McQuillan, A. J., “Raman Spectra of Pyridine Adsorbed at a Silver Electrode,” Chem. Phys. Lett, 26, 123, (1974), and Weaver, M. J., Farquharson, S., Tadayyoni, M. A., “Surface-enhancement factors for Raman scattering at silver electrodes. Role of adsorbate-surface interactions and electrode structure,” J. Chem. Phys., 82, 4867–4874 (1985)). Briefly, incident laser photons couple to free conducting electrons within the metal which, confined by the particle surface, collectively cause the electron cloud to resonate. The resulting surface plasmon field provides an efficient pathway for the transfer of energy to the molecular vibrational modes of a molecule within the field, and thus generates Raman photons.
The described phenomenon occurs however only if the following two conditions are satisfied: (1) that the free-electron absorption of the metal can be excited by light of wavelength between 250 and 2500 nanometers (nm), preferably in the form of laser beams; (2) that the metal employed is of the appropriate size (normally 5 to 1000 nm diameter particles, or a surface of equivalent morphology), and has optical properties necessary for generating a surface plasmon field. (Weaver, J. Chem. Phys., 82, 4867–4874 (1985), and Pettinger, J. Chem. Phys., 85, 7442–7451 (1986), supra).
Analyses for numerous chemicals and biochemicals by SERS has been demonstrated, but none of the foregoing techniques is capable of providing quantitative measurements with reproducible results.